In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Our Branch has generated a knock-in murine model for OI with a classical collagen mutation, and we are using this model to study disease pathogenesis and the skeletal matrix of OI, the effects of pharmacological therapies, and approaches to gene therapy.We are also continuing our clinical studies of children with types III and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for treatment of their condition. [unreadable] [unreadable] The Brtl mouse model for OI continues to be investigated to understand the pathological and cellular mechanism of OI. We first approached the secretion, matrix incorporation and interactions of collagen molecules with one and two mutant alpha 1 (I) chains. Collagen with no mutant chains or one mutant chain would be expected to comprise 25 and 50% of matrix collagen content; however we detected a decreased content of molecules with one mutant chain and an increase in the proportion with no mutant chains. We demonstrated selective cellular retention of molecules containing one mutant chain, compared to those with no or two mutant chains. In matrix, collagens were incorporated into matrix proportionately to their presence in media, pointing to the cell as the site of discrimination. Brtl fibroblasts had engorgement of the endoplasmic reticulum, suggesting that these cells were undergoing ER stress. The reactive -SH group in collagen with one mutant chain is exposed to solvent and may form aberrant S-S bonds with other intracellular or extracellular proteins.[unreadable] [unreadable] We also studied the basis for the phenotypic variability in Brtl, since this models the phenotypic variability seen in human alpha 1(I) mutations. Brtl has two discrete phenotypes; about 30% of the pups die within hours of birth, while the survivors have a moderately severe skeletal defect. We used microarray and 2D proteomics for complementary studies of calvarial bone from lethal and surviving Brtl pups. We found that Gadd153 expression was increased 2-3 fold in lethal Brtl mice in comparison to surviving and wt littermates. The increase in Gadd153 expression was detected only in bone tissue and was not found in skin or lung. Furthermore, increased Gadd153 was also present in calvarial protein extracts by Western blot. Gadd153 is a member of the C/EBP family that can pair with C/EBP beta or alpha to inhibit osteoblast differentiation or to promote cellular apoptosis, respectively. Conversely, the alpha B-crystallin chain was determined to be relatively increased in both expression (1.6 fold) and protein (doubled) in the non-lethal Brtl pups. alpha B-crystallin has a role opposite to that of Gadd153. It is a small heat shock protein, which is known to confer resistance to apoptosis. This data suggests that apoptosis is the key factor in the phenotypic variability of Brtl mice. We are currently exploring the dimerization partners of Gadd153 in Brtl osteoblasts in culture.[unreadable] [unreadable] To better understand the relationship of phenotype to genotype in human OI cases, the BEMB led an international consortium of connective tissue laboratories to assemble and analyze a database of structural mutations in type I collagen causing OI. The consortium assembled over 830 mutations, including 682 glycine substitutions and 150 splice site defects. Genotype-phenotype modelling revealed different functional relationships for each chain of type I collagen. Glycine substitutions in the alpha 1 (I) chain have a generally more severe outcome, with 36 percent of substitutioons resulting in a lethal phenotype. Substitutions by residues with charged or branched side chains have a lethal outcome in the majority of occurrences. Mutations in the amino quarter of the chain are non-lethal; even those involving residues with charged or branched side chains. We observed two stretches of exclusively lethal mutations in the carboxyl quarter of the chain. The two regions coincide with the Major Ligand Binding Regions (MLBR) of several ligands on the collagen monomer, including integrins and fibronection. Glycine substitutions in the alpha 2 (I)chain have a more moderate outcome on average, with less than 20 percent of occurrences resulting in lethality. Substitutions by residues with charged side chains are predominantly lethal, as in alpha 1 (I), but valine, with a branched side chain, is lethal in only 17 percent of occurrences in alpha 2 (I)(as compared with 73% in alpha 1 (I)). As for alpha 1 (I), occurrences in the amino third of the chain are non-lethal. Thereafter, the lethal mutations occur in eight clusters that are regularly spaced along the chain. The distribution of lethal mutations in alpha 2 (I) continues to follow the pattern we previously described in the Regional Model for this chain and correctly predict the phenotype of 86% of cases in alpha 2 (I). The lethal regions coincide with the binding regions for matrix proteoglycans on the collagen fibril. Finally, splice site mutations lead to mild OI in a minority of cases. Most splice site mutations, even those in invariant positions, lead to significant dysplasia, suggesting that use of an alternative donor or acceptor generates translatable products that can be incorporated into matrix. Most mutations that lead to simple exon skipping have a severe or lethal outcome. This modelling provides testable hypothesis for the mechanisms of OI.[unreadable] [unreadable] The BEMB undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both the primary skeletal gains (increased bone density and decreased fractures) and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. Children in the treatment group received pamidronate for 18-23 months. The treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area. However, the increment in vertebral BMD in the treatment group tapered off after one to two years of treatment. Furthermore, the treatment group did not experience a decrease in long-bone fractures. No functional effect was seen from bisphosphonate treatment. There was no significant change in ambulation level, lower-extremity strength or pain in children with OI treated with pamidronate. Some patients reported increased endurance or decreased back pain, but most reported no perceptible changes. Hence, the changes previously reported in these parameters appear to have been a placebo effect in the uncontrolled trials. The data from our controlled trial were in accord with three other controlled trials that were conducted in the same time frame. We are now recommending that treatment of children with types III and IV OI with pamidronate be limited to one to two (or at most three) years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial, using the dose from our first trial and a lower dose. This trial has now entered the data analysis phase; our hypothesis is that the children will gain a comparable benefit from the lower dose with decreased detrimental effects.